Since its cloning in 1995 and its identification (under this grant) as an unprecedented intramembrane aspartyl protease in 1999, Presenilin has been implicated in a remarkable array of signaling and regulatory events in all metazoans. PS was discovered through research on Alzheimer's disease, but it was soon shown to confer functions necessary for life, especially as the protease that enables Notch nuclear signaling. Thus, continuing to decipher the structure, functions, and protein and lipid regulators of PS is a priority for basic cell biology. At the same time, the invariant cerebral accumulation of amyloid beta-protein (AU) decades before symptoms of dementia has made PS/gamma-secretase a key target for mechanistic and therapeutic study in AD. Despite its pleiotropic role in biology, the protease's structure has only been resolved at 12 A (under this grant), and small molecules that can selectively inhibit its processing of APP are not yet validated. For all these reasons, six collaborators with deep experience in the study of Presenilin wish to apply a range of techniques in cell biology, genetics, chemistry, structural biology and animal modeling to tackle some of the thorniest questions in PS/gamma-secretase biology. Can one derive an atomic resolution structure of this 19- transmembrane complex? What is the cell biological mechanism of coordinated alpha-, beta- and gamma-secretase processing? How do certain synaptic proteins and membrane lipids regulate PS activity in neurons, affecting the crucial A(l42/4o ratio? Can one design drugs that are sufficiently potent yet selective to chronically inhibit gamma-secretase? Our group has carefully revised this application to address all of the thoughtful critiques the SEP offered. We propose numerous interrelated aims that incorporate three cross-cutting themes which unite our work. First, we will further confirm and extend our recent discovery of an endogenous complex of the alpha/beta/gamma-secretases (a sheddasome) that may mediate the efficient, sequential processing of APP - and presumably all gamma-substrates. Second, we will apply a unique FRET-based probe developed here to measure PS conformation in living neurons and learn if certain synaptotagmins we recently identified by proteomics as novel interactors of both PS1 and APP enable PS to change its conformation rapidly and reversibly in response to Ca2+ influx at the synapse, explaining the enhancement of AB production by neural activity. Third, we'll study novel gamma-secretase modulators and Notch-sparing inhibitors we've developed to define SARs for APP vs. Notch cleavage, identify the cognate binding sites, and assess their actions on other substrates, all with the goal of advancing one or more into preclinical development. In short, we are committed to applying novel approaches to elucidate the structure and function of gamma-secretase in health and disease.